Temporal Multiplexed Excitation for Miniaturized, Planar Fluorescence Activated Cell Sorting

ABSTRACT

A system for fluorescence activated cell sorting includes at least two excitation lasers and an objective that directs light from the at least two excitation lasers to a common point in an interrogation region of a fluidic channel. The fluidic channel directs a flow of a plurality of fluorescently labeled particles through the interrogation region. At least one modulator temporally multiplexes light from the at least two excitation lasers such that pulses of light from different lasers intersect the common point at different times. The system further includes at least one detector and at least one optical element that directs light emitted from the particles and transmitted through the objective to the at least one detector. The system may further include optics for generating and detecting side and forward scattered light. Methods for operating example systems to collect fluorescent, side scattered and forward scattered light are also described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/613,614, filed Jun. 5, 2017, which claims priority to U.S.Provisional Patent Application No. 62/346,206, filed Jun. 6, 2016. Theforegoing applications are incorporated herein by reference.

BACKGROUND

Unless otherwise indicated herein, the materials described in thissection are not prior art to the claims in this application and are notadmitted to be prior art by inclusion in this section.

Fluorescence activated cell sorting (FACS) is a technique used incytometry for measuring, sorting and enriching rare cells and particles,such as beads, from large heterogeneous populations. While FACS systemsoffer desirable multiplexing performance, they can be large andexpensive and are typically operated by specially trained staff. As aresult, FACS systems are used in only a relatively small number offacilities. To allow for more widespread availability, miniaturized FACSsystems (often called “μFACS”) have been developed. However, there arefew commercial systems to date and those that exist have limited numberof both detection and sorting channels compared to traditional FACS.

In one example μFACS system, the laser excitation light shares a commonpath with the flowing cells. This configuration can limit themicrofluidic geometry and involves specialized coatings on the channelto permit optical waveguiding. Such coatings can make the chips morecostly to fabricate and may not be suitable for all biological samples.Further, this example system only allows for a single excitationwavelength, which is not desirable if more than about ten fluorescentmarkers are to be identified. Another example μFACS system offers morechannels with up to 4 excitation lasers and 8 fluorescent channels(along with two scatter channels), but employs a cuvette such that theexcitation lasers are perpendicular to the collection path. Althoughthis approach may have the advantage that the side scatter channel couldbe efficiently collected, it may be desirable to use widely available,planar microfluidic chips instead of cuvettes. In addition, thisapproach uses a large number of detectors, such as photomultiplier tubes(PMTs).

Accordingly, there is a need for systems that are compatible withexisting FACS protocols, that are in a planar geometry such as amicrofluidic chip, and that can employ a number of simultaneousfluorescent markers and emission channels on the order of what ispossible with traditional FACS systems.

SUMMARY

The present invention is directed to μFACS systems and related methods,in particular, μFACS systems having at least two temporally multiplexedexcitation lasers.

In one aspect, a system is provided, comprising: (a) at least twoexcitation lasers; (b) an objective that directs light from the at leasttwo excitation lasers to a common point in an interrogation region of afluidic channel, wherein the fluidic channel directs a flow of aplurality of fluorescently labeled particles through the interrogationregion; (c) at least one modulator that temporally multiplexes lightfrom the at least two excitation lasers such that pulses of light fromdifferent lasers intersect the common point in the interrogation regionof the fluidic channel at different times; (d) at least one detector;and (e) at least one optical element optically coupled to the objectiveand the at least one detector to direct light emitted from the pluralityof fluorescently labeled particles and transmitted through the objectiveto the at least one detector.

In another aspect, a method is provided, comprising the steps of: (a)moving a plurality of fluorescently labeled particles through a fluidicchannel comprising an interrogation region, wherein the plurality offluorescently labeled particles move through the interrogation region ata flow speed; (b) directing light from at least two excitation lasersthrough an objective to a common point in the interrogation region ofthe fluidic channel; (c) temporally multiplexing light from the at leasttwo excitation lasers such that pulses of light from different lasersintersect the common point in the interrogation region of the fluidicchannel at different times; (d) receiving, by at least one detector,light emitted from each of the plurality of fluorescently labeledparticles and transmitted through the objective; and (d) generating, bythe at least one detector, a fluorescence signal indicative of intensityof light emitted from each of the plurality of fluorescently labeledparticles as the particles move through the interrogation region.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic view of an example optical architecture of anembodiment of a μFACS system.

FIG. 2 is a top view of an example interrogation region of a fluidicchannel, and illustrating a side scatter beam spot and interrogationbeam spot generated by the example μFACS system of FIG. 1.

FIG. 3A is a side view of an example interrogation region, illustratingside scatter light generated as a particle passes through a side scatterbeam generated by the example μFACS system of FIG. 1.

FIG. 3B is a side view of an example interrogation region, illustratingforward scatter and fluorescent light generated as a particle passesthrough an interrogation light beam generated by the example μFACSsystem of FIG. 1.

FIG. 4 is a flow chart of an example method.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying figures, which form a part hereof. In the figures, similarsymbols typically identify similar components, unless context dictatesotherwise. The illustrative embodiments described in the detaileddescription, figures, and claims are not meant to be limiting. Otherembodiments may be utilized, and other changes may be made, withoutdeparting from the scope of the subject matter presented herein. It willbe readily understood that the aspects of the present disclosure, asgenerally described herein, and illustrated in the figures, can bearranged, substituted, combined, separated, and designed in a widevariety of different configurations, all of which are explicitlycontemplated herein.

Overview

Flow cytometry is an analytical technique used to measure and analyzethe physical and chemical characteristics of individual particles, suchas cells, as they flow in a fluid stream through a beam of light. Theproperties measured can include the relative size, relative granularityor internal complexity, and relative fluorescence intensity of eachindividual particle. Typically, cell components are fluorescentlylabelled and then excited by the laser to emit light at variouswavelengths. The emitted light is received by one or more detectors, andanalyzed, for example, based on how the cell or particle scattersincident laser light and emits fluorescence. FACS is a particular formof flow cytometry that enables a mixture of different particles or cellsto be sorted one by one into one or more containers according to theirspecific light scattering and fluorescence characteristics.

Traditional FACS systems, while being generally commercially availableand offering desirable capabilities, can be undesirably large andexpensive. Further, while there has been an effort to developminiaturized FACS systems (often described as μFACS systems), very fewcommercial μFACS systems currently exist.

In the present disclosure, an example multi-laser μFACS system havingtwo or more temporally multiplexed individual excitation lasers sharingthe same optical path is described. With this orientation, theexcitation laser spots are coincident on the fluidic channel. Theexample μFACS system may have a reduced number of detectors, as comparedto traditional FACS systems. In some embodiments, the μFACS system iscompatible for use with a planar microfluidic chip.

Example μFACS Optical Systems

FIG. 1 illustrates a schematic of an example optical architecture of aμFACS system 100 having at least two excitation lasers. In this example,three individual excitation lasers 102, 104, 106 are provided. The useof more than three excitation lasers is contemplated. Light from theexcitation lasers 102, 104, 106 is directed through an objective 120 toan interrogation region 122 of a fluidic channel 116. The fluidicchannel, which may be defined in a planar microfluidic chip 118, directsa flow of a plurality of fluorescently labeled particles through theinterrogation region 122. In the example shown in FIG. 1, the fluidicchannel 116 is positioned below the objective 120, such that excitationbeams 108, 110, 112 from each of the excitation lasers are incident onthe fluidic channel in a plane substantially perpendicular to thedirection of fluid flow (A) in the channel.

One or more cylindrical lenses 114A, 114B, 114C may be provided for eachlaser to shape each of the excitation beams 108, 110, 112 so that thedesired beam profile is delivered to the fluidic channel 116 positionedbelow the microscope objective 120. In flow cytometry applications, itcan be desirable to shape the excitation lasers such that they have anelliptical profile and orient the laser spots so that the longer axis ofeach ellipsis is perpendicular to the direction of flow in the fluidicchannel.

In this embodiment of system 100, the individual excitation lasers 102,104, 106 are spectrally multiplexed with dichroics Da, Db such that thebeams overlap with each other and share a common optical path throughthe objective 120. Accordingly, each of the excitation beams 108, 110,112 is coincident at the same point of the fluidic channel. Modulators150, 152, 154 are provided for sequentially pulsing the individualdrivers 150, 151, 152 such that the pulses from the separate lasers aretemporally multiplexed. With this configuration, pulses from each of thelasers 102, 104, 106 will intersect the fluidic channel 116 at the samepoint, but at different times. In the example shown in FIG. 1, themodulators 150, 152, 154 comprise drivers that modulate the excitationbeams 108, 110, 112 by altering the drive current supplied to each ofthe excitation lasers 102, 104, 106. Alternatively, because somesolid-state lasers are not well-suited for direct current modulation,the modulators 150, 152, 154 can be provided as external modulators (notshown), such as gates, for generating the desired pulses.

FIG. 2 is a top view of a portion of μFACS system 100. A samplecontaining a plurality of fluorescently labelled particles (not shown),such as cells, is introduced into the fluidic channel 116. The system100 may be designed to detect a plurality of different fluorophores by,for example, using lasers emitting at different wavelengths. Forexample, different types of particles or cells may each be labelled withdifferent fluorophores, thereby allowing each type of particle or cellto be identified or categorized. The lasers 102, 104, 106 may beselected to emit at a suitable color to excite the fluorophoresselected. Individual particle 200, labeled with one or more fluorophores202, move in the fluidic channel 116 in the direction of fluid flow (A).The fluid flows at a particular flow speed, which may be known and maybe manually or automatically controlled. The particle 200 isinterrogated as it passes through the coincident laser spots 109, 111,113 in the interrogation region 122. Also shown is a side scatter beamspot 139, located upstream of the interrogation region 122, with respectto the direction of fluid flow (A), which will be discussed in furtherdetail below.

Fluorescence 130 emitted by each particle as it passes through thecoincident laser spots 109, 111, 113 is imaged through the objective 120and tube lens 124 (if needed) onto one or more detectors 126, such asPMTs, for each of four emission channels I-IV. The amount of fluorescentlight emitted can be correlated with the quantity of a particular cellor particle present in the sample. In the example shown in FIG. 1, thefluorescence emission is spectrally separated with a series of dichroicsD1, D2, D3 and D4 into the respective emission channels I-IV. Thus, foreach emission wavelength, only one single-pixel detector, such as suchas a standard PMT, may be provided. By temporally multiplexing theexcitation lasers 108, 110, 112, the fluorescence emission 130associated with each laser will be temporally separated at each of thedetectors 126. The fluorescence detectors 126 measure the amplitudes ofthe fluorescent signals generated by the different fluorescent markersas they move through the interrogation region 122. Numerical values aregenerated based on pulse heights (amplitudes) measured by each of thevarious detectors 126. The resulting signals can be input into aprocessor (not shown) and used to create histograms corresponding to thedetected events.

The characteristics of the driving pulse for the excitation lasers 102,104, 106 can be selected based on one or more factors including, but notlimited to, the fluid flow speed, the size of the particles underinterrogation, the spot size of each excitation laser on the fluidicchannel, the number of emission channels, emission time of the utilizedfluorophores, and the response time of detectors 126. The overall periodof the pulses (the time it takes to cycle through all of the individuallasers) can be short enough such that the particles being interrogatedare equally sampled by each excitation laser. The length of each pulseand the spacing between pulses of each laser can dictate the overallperiod of the pulses. However, the interval between the individual laserpulses can be long enough to account for the emission time of thefluorophores and the response time of the detectors and the downstreamelectronics, so that emission generated from each of the excitationlasers is temporally separated at each detector.

In one example, assuming that the maximum flow speed within the fluidicchannel 116 is 1000 mm/s and the spot size of an excitation laser is 10μm wide, a particle will traverse the spot size plane in 10 μs. Withfour excitation lasers, 2 μs pulses with 500 ns spacing can ensuretemporal separation of emission at the detectors. A wider spot sizecould permit longer pulses, longer spacing between the pulses, or both.In general, the fluorescence lifetime of fluorophores used in FACS istypically on the order of a few nanoseconds, so significant spillage ofsignal from one temporal slice to another is not likely. Alternately, atthe same flow speed, 1 μs pulses could be used to provide oversamplingof each particle. In some cases, it may be desirable to excite aparticle several times with each laser as it passes through theinterrogation space. Further, if the flow speed is known, the temporalmultiplexing could be optimized to maximize the collected signal. Theorder of the lasers could also be optimized so that lasers with minimalspectral overlap are turned on sequentially.

In the illustrated embodiment of system 100, the excitation path sharesa common path with the collection path, as they are both coupled throughthe objective 120. An optical element 128 directs light that is emittedfrom the plurality of fluorescently labeled particles after it passesthrough the objective 120 to the detectors 126. In one example, theoptical element 128 is provided as a dichroic D1. The dichroic D1 can,in one example, have narrow transmission peaks for the excitation laserwavelengths and reflects all other wavelengths to the fluorescencedetectors 126. For simplicity, emission channels II-IV are illustratedwith broken lines. Dichroics D2, D3 and D4 direct each emittedfluorescence wavelength to a detector 126 for each respective emissionchannel. A bandpass filter F1, F2, F3 and F4 may also be provided foreach of emission channels I-IV. While FIG. 1 illustrates dichroic D1transmitting the excitation laser wavelengths and reflecting theemission wavelengths, this could alternatively be switched such that D1reflects the excitation laser wavelengths and transmits the emissionwavelengths. The remaining optics in the system can be reconfigured asnecessary.

Alternatively, the optical element 128 may be provided as a dot mirror,in which the excitation laser beams 108, 110, 112 are reflected by smallmirrors in a window (called “dot optic”). Since the emitted fluorescencelight will occupy the entire back aperture of the objective 120 and aconsiderable amount of the area of the dot optic, the percentage ofemitted light lost in the collection path due to the small mirrors issmall. In the three laser system illustrated in FIG. 1, three smallmirrors would be required. One possible advantage of this embodiment isthat the dot mirror may be less costly to produce and may provide betterperformance than a multi-band dichroic, such as D1. Alternatively, theoptical element 128 may be a dot optic configured to transmit theexcitation laser beams 108, 110, 112 through small apertures in amirror. In either case, because all of the excitation beams arespatially coincident, only a single small mirror or aperture would beneeded, regardless of the number of excitation lasers.

In addition to fluorescence emission, forward and side scattered lightmay also be detected and measured by the system 100. A forward scatterdetector 134 and a side scatter detector 140 can generate electricalsignals corresponding to detected events as the cells or particles aredirected through the fluid channel 116. In one example, shown in FIG. 1,one of the incident laser beams 108, 110, 112 is used for the forwardscattering channel. Forward scattered light 132 is scattered through thefluidic channel 116 and is collected by the detector 134. A bandpassfilter 135 and one or more tube lenses 136 may also be provided in theoptical path.

For side scatter measurements, the same or a different one of theincident laser beams 108, 110, 112 can have its power partially pickedoff with a beamsplitter BS to provide a side scatter beam 138. A mirrorM1 directs the side scatter beam 138 to the fluidic channel 116, andside scattered light 144 is diffusely reflected by particles in thefluidic channel 116. In one implementation (shown in FIG. 1), the sidescatter beam 138 is directed to an area of the fluidic channel 116 thatis located upstream of the interrogation region 122. In thatimplementation, the side scattered light 144 is detected by the detector140 through an optical path that is separate from the objective 120. Oneor more tube lenses 142 may also be provided in the optical path. In analternative implementation (not shown), the side scatter beam 138 isdirected to the interrogation region 122. In this alternativeimplementation, the side scattered light 144 is imaged through theobjective and detected by the detector 140.

The laser path used for side scatter shown in FIG. 1 (side scatter beam138 directed to the fluidic channel 116 upstream of the interrogationregion 122) could also be used for forward scatter measurements insteadof using one of the incident laser beams through the objective 120. Oneadvantage of this alternative architecture is that the system 100 may beable to detect particles more quickly if additional synchronizationsteps are needed, as both the side and forward scatter measurements aretaken prior to the fluorescence interrogation area. In an additionalalternative embodiment, side scatter could be measured below the fluidicchannel. This configuration may be advantageous as there is more spaceunderneath the channel and it may generate fewer Fresnel reflections.

The objective 120 may also be used in brightfield detection (not shown).This may require an appropriate substitution of the dichroic and anincoherent light source. Brightfield detection may be used to observethe beam spots 109, 111, 113 in the field of view in order to ensureproper alignment of the spots 109, 111, 113 with the fluidic channel116.

FIG. 3A and FIG. 3B illustrate a side view of a portion of the fluidicchannel 116 of the system 100. In the example shown in FIG. 3A, sidescattered light 144 is emitted from the fluidic channel 116 when aparticle 200 passes through the side scatter beam 138. In the exampleshown in FIG. 3A, the system 100 is configured such that the sidescatter beam 138 intersects the fluidic channel 116 at a positionupstream of the position at which the excitation laser beams 108, 110,112 intersect the channel 116. Alternatively, the side scatter beam 138could intersect the fluidic channel 116 within the interrogation region122 so that the side scattered light 144 is imaged through the objective120.

Forward scattered light 132, in the example shown in FIG. 3B, istransmitted through the fluidic channel 116 when a particle 200 passesthrough the coincident excitation beams in the interrogation region 122.Fluorescence 130 may be generated, depending on the particularfluorophore(s), as a particle 200 moves through the excitation beams108, 110, 112.

In order to process the output of the system 100, and ensure that eachof the collected signals are assigned to the correct particle in asample, the side scatter, forward scatter and fluorescence signals fromthe various laser spots 109, 111, 113 can be synchronized. To do this,the flow speed of the fluid stream in the fluidic channel 116 isdetermined. In one embodiment, the flow speed of the fluidic channel maybe externally controlled, and therefore has a known value. The system100 may combine each of the scatter and emission signals into a singleflow event, provide closed-loop flow speed regulation and preciselysynchronize actuation of a deflection system, which is used to sort aparticle under interrogation into a capture channel of interest.

The present μFACS system 100 may provide several advantages overexisting approaches. First, this approach is compatible with planarmicrofluidic chips. Second, this approach may allow for a more compactoptical setup, while providing as many illumination inputs and outputsas existing FACS systems. Third, this setup may have a simpler method ofalignment as all excitation and emission pass through a singleelement—the objective 120. Fourth, this approach may allow for a moreeconomical detection approach since temporal multiplexing permits theuse of a single pixel detector for each emission channel, therebyallowing for more channels with fewer detectors.

Example Method

A flowchart of an example method 400 for operating a μFACS system havingat least two lasers to collect one or more of fluorescent, side scatteror forward scatter light, is shown in FIG. 4. In a first step (402), aplurality of fluorescently labeled particles is moved through a fluidicchannel, including an interrogation region, of a μFACS system, such assystem 100. The plurality of fluorescently labeled particles movesthrough the interrogation region at a flow speed, which may be known ormeasured during operation of the system. The plurality of particles maybe obtained from a sample source, such as a microtiter plate, by, forexample, a probe in fluid communication with the fluidic channel. Insome examples, the particles are drawn from the sample source into thefluidic channel by means of a pump, such as a peristaltic pump. Thelight from at least two excitation lasers is directed through theobjective to a common point in the interrogation region of the fluidicchannel. (404). Further, the light from the at least two excitationlasers are temporally multiplexed such that pulses of light fromdifferent lasers intersect the common point in the interrogation regionof the fluidic channel at different times. (406). Light emitted fromeach of the plurality of fluorescently labeled particles passes throughthe objective, and is received by at least one detector. (408). The atleast one detector generates a fluorescence signal corresponding to theintensity of light emitted from each of the plurality of fluorescentlylabeled particles, as the particles move through the interrogationregion. (410).

The method may also include steps for collecting side and forwardscatter light with a μFACS system having at least two lasers, such assystem 100. In some examples, a portion of the light from one of the atleast two lasers is directed to a location in the fluidic channel forside scatter measurements. The location could be, for example, withinthe interrogation region or upstream of the interrogation region withrespect to the direction of flow in the fluidic channel. At least oneside scatter detector receives side scattered light from each of theplurality of fluorescently labeled particles and generates a sidescattered signal. The side scattered light is, in some examples, emittedfrom within the fluidic channel. In some examples, at least one forwardscatter detector receives forward scattered light from each of theplurality of fluorescently labeled particles and generates a forwardscattered signal. In some examples, the forward scattered light istransmitted through the fluidic channel.

The generated fluorescence, forward scatter, and side scatter signalsattributed to a single particle of the plurality of particles can alsobe synchronized by the system 100. In some examples, the various signalsmay be synchronized based, at least in part, on the flow speed of theplurality of particles in the fluidic channel. If, for example, the flowspeed is known or determined, the system may identify certain events inthe fluorescence, forward scatter, and side scatter signals as beingattributable to a single particle. The flow speed may be, in someexamples, be set and controlled by a pump. Alternatively, the flow speedmay be measured or determined.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopebeing indicated by the following claims.

1. A system comprising: at least two excitation lasers; an objectivethat directs light from the at least two excitation lasers to a commonpoint in an interrogation region of a fluidic channel, wherein thefluidic channel directs a flow of a plurality of fluorescently labeledparticles through the interrogation region; at least one modulator thattemporally multiplexes light from the at least two excitation laserssuch that pulses of light from different lasers intersect the commonpoint in the interrogation region of the fluidic channel at differenttimes; at least one detector; and at least one optical element opticallycoupled to the objective and the at least one detector to direct lightemitted from the plurality of fluorescently labeled particles andtransmitted through the objective to the at least one detector.
 2. Thesystem of claim 1, wherein the fluidic channel is defined in a planarmicrofluidic chip.
 3. The system of claim 1, wherein the at least onemodulator sequentially pulses the at least two excitation lasers.
 4. Thesystem of claim 1, wherein the at least one detector comprises at leastone single pixel detector.
 5. The system of claim 1, wherein the atleast one detector comprises a single pixel detector for each wavelengthof light emitted by the plurality of fluorescently-labeled particles. 6.The system of claim 1, wherein the at least one optical elementtransmits incident light from each of the at least two excitation lasersand reflects light emitted by the plurality of fluorescently-labeledparticles.
 7. The system of claim 6, wherein the at least one opticalelement comprises a dichroic having narrow transmission peaks for eachwavelength of light incident by the at least two excitation lasers. 8.The system of claim 6, wherein the at least one optical elementcomprises a dot optic.
 9. The system of claim 1, wherein the at leastone optical element reflects incident light from each of the at leasttwo excitation lasers and transmits light emitted by the plurality offluorescently-labeled particles.
 10. The system of claim 9, wherein theat least one optical element comprises a dichroic having narrowtransmission peaks for each wavelength of light emitted by the pluralityof fluorescently-labeled particles.
 11. The system of claim 9, whereinthe at least one optical element comprises a dot mirror.
 12. The systemof claim 1, wherein the at least two excitation lasers comprises atleast three excitation lasers.
 13. The system of claim 1, furthercomprising a detector for detecting forward scattered light transmittedthrough the fluidic channel.
 14. The system of claim 1 furthercomprising at least one detector for detecting side scattered lightreflected from the fluidic channel.
 15. The system of claim 14, furthercomprising at least one optical element for directing a portion of thelight from one of the at least two lasers to the fluidic channel at aposition upstream of the interrogation region with respect to thedirection of flow of the plurality of fluorescently labeled particles inthe fluidic channel.
 16. A method comprising: moving a plurality offluorescently labeled particles through a fluidic channel comprising aninterrogation region, wherein the plurality of fluorescently labeledparticles move through the interrogation region at a flow speed;directing light from at least two excitation lasers through an objectiveto a common point in the interrogation region of the fluidic channel;temporally multiplexing light from the at least two excitation laserssuch that pulses of light from different lasers intersect the commonpoint in the interrogation region of the fluidic channel at differenttimes; receiving, by at least one detector, light emitted from each ofthe plurality of fluorescently labeled particles and transmitted throughthe objective; and generating, by the at least one detector, afluorescence signal indicative of intensity of light emitted from eachof the plurality of fluorescently labeled particles as the particlesmove through the interrogation region.
 17. The method according to claim16, further comprising: directing a portion of the light from one of theat least two lasers to the fluidic channel at a position upstream of theinterrogation region with respect to the direction of flow in thefluidic channel; and receiving, by at least one side scatter detector,side scattered light from each of the plurality of fluorescently labeledparticles; generating a side scattered signal by the at least one sidescatter detector; receiving, by at least one forward scatter detector,forward scattered light from each of the plurality of fluorescentlylabeled particles; and generating a forward scatter signal by the atleast one forward-scatter detector.
 18. The method of claim 17, whereinthe side scattered light is emitted from within the fluidic channel. 19.The method of claim 17, wherein the forward scattered light istransmitted through the fluidic channel.
 20. The method of claim 17,further comprising: synchronizing the fluorescence signal, forwardscatter signal and side scatter signal attributed to a single particleof the plurality of particles based, at least in part, on the flowspeed.